Vol. $3, No. 3, March 1968
SYNTHESIS OF SOME QUINAZOLINE NUCLEOSIDES 1219
The Synthesis of Some Quinazoline Nucleosides’ MASONG. STOUT AND ROLAND K. ROBINS Department of Chemistry, University of Utah, Salt Lake City, Utah 8411.9 Received August 31, 1967 The synthesis of l-(p-~-ribofuranosyl)-2,4quinazolinedione (IV) has been accomplished in above 80% yield by treatment of 2,4bis(trirnethylsilyloxy)quina~olinewith 2,3,5-tri-O-benzoyl-~-ribofuranosyl bromide followed by removal of the benzoyl groups with methanolic ammonia or sodium methoxide. Proof of the &configuration was obtained by stepwise conversion of IV into 02, 5’-anhydro-l-(2’, 3‘-O-isopropylidene-p~-ribofuranosyl)-4quina~olone (XI). The site of sugar attachment was established as NI by methylation of IV followed by acidic hydrolysis of the product to yield 3-methyl-2,Cquinazolinedione. This assignment was confirmed by ultraviolet and infrared absorption data. Treatment of 1-(2’,3’,5’-tri-0-benzoyl-p-~-ribofuranosyl)-2,4-quinazolinedionewith phosphorus pentasulfide provided the 4-thio derivative (V) which upon reaction with methanolic ammonia a t 100” resulted in replacement of the 4-thio group with concomitant (VII). Compounds IV and VI1 may debenzoylation to yield 4amino-l-(~-~-ribofuranosy~)-2-quinazo~one be regsrded as 5,6-benzouridine and 5,6-benzocytidine, respectively, with a fused planar aromatic system. The possible biochemical significance of greater electron interaction in the stacking of heterocyclic bases is (XIX) and discussed. Using similar procedures, 1-(2’-deoxy-p-~-r~bofuranosyl)-2,Pqu~na~ol~ned~one 4-am~no-l-(2’-deoxy-p-~-r~bofuranosy~)-2-qu~nazo~one (XXI) have also been prepared. l-(B-D-Ribofuranosyl)-2,4-quinazolinedione (IV) has successfully been converted into 02,2’-anhydro-l-(p-~-arabinofuranosyl)-4quinazolone (XII) which yielded 1-(~-~-arabinofuranosy~)-2,4quinazo~inedione (XIII) upon ring opening with dilute sodium hydroxide. Acetylation of XI11 followed by thiation and treatment with (XVI). The reaction procedure methanolic ammonia gave 4amino-l-(~-~-arabinofuranosyl)-2-quinazo~one for nucleoside formation in such good yields would indicate that this procedure might well be the method of choice for nucleoside synthesis with other unusual heterocyclic systems.
with sodium iodide gave the 5’-iodo compound which The use of trimethylsilyl derivatives of nitrogen was not isolated but cyclized directly to 02,5’-anhydroheterocycles in nucleoside synthesis was first intro1-(2’,3’-O-isopropylidene-/3-~ribofuranosyl)-4quinaz~ duced by Birkofer, et a1.,2 in a novel synthesis of lone (XI) with silver acetate in absolute methanol. 3-ribofuranosyluric acid. The use of 2,4-bis(triThe cyclonucleoside X I was also prepared directly methylsilyloxy) pyrimidine in a fusion procedure with from X by the action of potassium t-butoxide in 2,3,5-tri-O-benzoylribofuranosylchloride was emdimethylformamide at 100”for 1.5 hr. ployed by Nishimura, Shimizu, and Iwai3 to give The fact that X I was actually a cyclonucleoside uridine in 35% yield. Initial attempts to condense was established by treatment of XI with liquid amtrimethylsilyloxypyrimidines and acetohalo sugars dimonia which gave 2-amino-1-(2’,3’-0-isopropylidene rectly in boiling benzene were unsu~cessful.~The use /3-~-ribofuranosy~)-4-quinazo~one. All attempts to of polar solventss and milder reaction conditions5t6has remove the isopropylidene group resulted in conbeen more successful. In the present study it was comitant hydrolysis of the nucleoside and 2-amino-4desired to attach the D-ribofuranosyl ring to position 1 quinazolone was isolated instead of 2-arnino-l-(P-Dof 2,4-quinazolinedione (I). Treatment of I with triribofuranosyl)-4-quinazolone. methylchlorosilane in the presence of toluene and triThe position of attachment of D-ribose was deterethylamine gave 83% yield of distilled colorless oil, 2,4mined in the following manner. A comparison of the bis(trimethylsily1oxy)quinazoline (11) (see Scheme I). ultraviolet absorption spectrum of IV with that of Pmr spectra indicated the presence of six methyl 3-methyl-2,4-q~inazolinedione~ and l-methyl-2,4quingroups. Treatment of I1 with freshly prepared 2,3,5azoline,8 respectively, showed that a t a pH of 1 or in tri-0-benzoylribofuranosyl bromide in dry acetonitrile neutral solution (see Table I) l-@-D-ribofuranosyl)for 2 days a t room temperature gave 90% yield of crystalline 1-(2‘,3‘,5’-tri-0-benzoy1-/3-~-ribofuranosy~)- 2,4-quinazolinedione (IV) and the 1-methyl- and 3-methyl-2,4-quinazolinediones possessed very similar 2,4-quinazolinedione (111). Treatment of I11 in a spectra with a A,, in the region of 306-312 mp. refluxing solution of sodium methoxide in methanol However at pH 11, 3-methyl-2,4-quinazolinedione exremoved the benzoyl groups to provide an 88% yield of 326 mp while of crystalline 1-(/3-~-ribofuranosyi)-2,4-quinazo~ine-hibited a bathochromic shift t o A, a t pH 11, l-methyl-2,4-quinazolinedione and the dione (IV). Proof of the structural assignment as /3 nucleoside IV exhibited no such shift (Table I). was obtained by the stepwise conversion of IV to 02,5‘-anhydro-1-(2‘,3‘-O-isopropylidene-p-~-ribofuran- Additional evidence for assignment of the sugar moiety to the 1 position was obtained by methylation osyl)-4-quinazolone (XI) (see Scheme 11). The nuof IV with methyl iodide in N,N-dimethylformamide cleoside IV was converted to 1-(2’,3’-0-isopropylidenein the presence of potassium carbonate. The crude /3-~-ribofuranosyl)-2,4-quinazolinedione (IX) and then 3-methyl- 1- (P-D-ribofuranosyl)-Z, 4-quinazolinedione to the ij’-O-mesyl derivative (X). Treatment of X was hydrolyzed with 10% hydrochloric acid to yield (1) This research was supported by Research Grant GB-5446 from the 3-methyl-2,4-quinazolinedione which was identical with Molecular Biology Division of the National Science Foundation. an authentic sample’ as judged by uv spectra, ir (2) L. Birkofer, A. R. Ritter, H. P. Kulthau, Anpcw. Chcm., 76, 209 spectra, and mixture melting point data. (1963). (3) T. Nishimura, B. Shimiau, and I. Iwai, Chem. Pharm. Bull. (Tokyo), Synthesis of the cytidine analog, 4-amino-l-(/3-~11, 1470 (1963).
(4) T. Nishimura and I. Iwai, i b i d . . 12,357 (1964). (5) E. Wittenberg, Z. Chem.. 4, 303 (1964). ( 6 ) K. J. Ryan, E. M . Acton, and L. Goodman, J . O w . Chem., Si, 1181 (1966).
(7) R. P. Steiger and E. C. Wagner, ibid.. 18, 1427 (1953). ( 8 ) A. Chatterjee and S. G. Majumdar. J . Amer. Chem. SOC.,76. 2459 (1954).
1220
STOUTAND ROBINS SCHEMB
0
The Journal of Organic Chemistry
I OSi(CH&
SCHEME I1 0
OH OH
lv 9
XI
O 2
Ix
l
e
X
mixture of VI1 contaminated with IV and in general was not as useful as the thiation route. Preparation of 1-(/3-~-arabinofuranosy1)-2,4-quinazolinedione (XIII) was accomplished in two steps from the uridine analog IV (see Scheme 111). The method of Nichol and Hampton’O for inversion of the 2‘-hydroxyl was employed. Treatment of IV with diphenyl carbonate and sodium bicarbonate in N,Ndimethylformamide gave O2,2’-anhydro-1-(/3-~-arabinofuranosyl)-4quinazolone (XII) in 90% yield. Treatment of XI1 with dilute sodium hydroxide on the steam bath gave l-(P-~-arabinofuranosyl)-2,4 OH OH quinazolinedione XI11 in quantitative yield. Acid hydrolysis of XI11 gave D-arabinose which was charVI1 acterized by chromatography. Acetylation of XI11 gave 1-(2’,3’,5’-tri-O-acetyl-@-~-arabinofuranosyl)-2,4-quinazolined~one (XIV). ribofuranosyl)-2-quinazolone (VII) , was accomplished in two steps by treatment of 1-(2’,3’,5’-tri-O-benaoyl-@- Treatment of XIV with phosphorus pentasulfide in pyridine gave 4-thio-1-(2’,3’,5’-tri-O-acetyl-/3-~-ara~-ribofuranosyl)-2,4-quinazolinedione (111) with phosbinofuranosyl)-2-quinazolone(XV) which was in turn phorus pentasulfide in pyridine to give a 93% yield of 4-thi~l-(2’,3’,5’-tri-O-benzoyl-@-~-ribofuranosyl)-2- treated with methanolic ammonia at 100” to give a good yield of 4-amino-l-(/3-~-arabinofuranosyl)-2quinazolone (V), and then reaction of V with methquinazolone (XVI), the benzo analog of “cytosine anolic ammonia a t 100’ gave simultaneous deblocking and replacement of the 4-thio group to provide 4- arabinoside.” For the synthesis of 1-(2’-deoxy-/3-~-ribofuranosyl)amino-1-(~-~-ribofuranosyl)-2-quinazo~one (VII), in 2,4quinazolinedione (XIX) , 2,4-bis( trimethylsilyl82% yield. This general method for the introduction 0xy)quinazoline (11) was treated with l-chloro-3,5-diof an amino group was first utilized in nucleoside chemO-p-tolyl-2-deoxyribofuranose11in acetonitrile (see istry-by Fox, et aLB A second route to VI1 involved the treatment of 1-(2’,3‘,5’-tri-0-beneoyl-/?-~-ribo- Scheme IV). An anomeric mixture of nucleosides was obtained which was separated on an alumina furanosyl)-2,4-quinazolinedione(111) with phosphorus column. A 25% yield of pure 1-(3’,5’-di-O-p-tolyl-Poxychloride and N,N-diethylaniline, to give the pre~-2’-deoxyribofuranosyl)-2,4quinazolinedione (XVIII) sumed 4-chloro intermediate VI which was not isolated was obtained plus an additional 35% of mixed anobut treated directly with methanolic ammonia to give mers. Deblocking XVIII with refluxing sodium 4-amino-l-(/3-~-ribofuranosyl)-2-quinazo~one (VII) in methoxide-methanol gave 1-(2’-deoxy-P-~-ribofuran52% yield. This latter procedure, however, gave a osyl)-2,4-quinazolinedione X I X in 69% yield. Treat-
Hot$
(9) J. J. Fox, D. Von Praag, I. Wempen, I. L. Doerr, L. Cheong, J. E. Knoll, M. L. Eidinoff, A. Bendich, and G. B. Brown, J . Amur. Chsm. Soc., 81,178 (1959).
(10) A. Hempton and A. W. Nichol, Biochemistry, I,2078 (1968). (11) M. Hoffer, Chem. Bcr., 93, 2777 (1980).
SYNTHESIS OF SOME QUINAZOLINE NUCLEOSIDES1221
Vol. 33, No. 3, March 1968
TABLEI ULTRAVIOLET ABSORPTION SPECTRAOF VARIOUSQUINAZOLINE NUCLEOSIDES -Xmu
pHll Xmax
I
309 240 (infl)
w 310 260 (in@
IV
306
306
VI11
355 325 (sh) 310 (sh) 270 239
348 274
VI1
326 258
3 17
XIX
307
307
XXI
326 258
316
XI1
309 (infl) 299.5
309 (infl) 300
(4,300) (5,200)
XI11
308
307
(4,300)
XVI
328 257
318
(4,900)
IX
306
307 356 (infl)
X
305 240
306 256
XI
304
307 (infl) 301
309 240 (infl)
326 266
3-N-Methyl-2,4-quinazolinedionea
309 240 (infl)
326 266
1-N-Met hyl-2,4-quina~olinedione~
3 12 242 (infl)
3 13
mp
a
See ref 7.
* See ref
---
EtOH
(4 500)
309 298
(4,600) (5,500)
8.
ment of XVIII with phosphorus pentasulfide in pyridine gave X X which in turn gave 4-amino-1-(2’deoxy-fl-D-ribofuranosyl)-%quinazolone (XXI) with methanolic ammonia. Although no chemical evidence was obtained, the anomeric configuration of X I X (and therefore XXI) was tentatively assigned as (3 on the basis of the pseudo triplet, J = 7.5 cps and peak width was 15.0 cps, due to the anomeric proton.12 The ultraviolet absorption spectral data for various quinazoline nucleosides are given in Table
I. The spatial limitations for the enzymatic incorporation of a substituted pyrimidine nucleoside into nucleic acid have not been defined although pyrimidines containing groups as large as iodine a t the 5 position have been incorporated.’a In this regard it may be noted that b“iso”-adenosine diphosphate (12) M. J. Robins and R. K. Robins,J . Amer. Chcm. Sw., 87,4934 (1965). (13) W. H. Prusoff, J. J. Jaffe. and € Gunther, I. Bioohem. Phormacd., 8 , 110 (1959).
is polymerized by azotobacter polynucleotide phosphorylase to yield 3-“iso”-polyadenylic acid.l4 I n the present study the fusion of a benzene ring at the pyrimidine positions 5 and 6 results in addition of an aromatic ring in the same plane as the pyrimidine moiety. These derivatives should provide very interesting nucleosides for biochemical study. If the corresponding nucleotides are not incorporated into nucleic acids due to the presence of the benzene ring it is quite possible that these nucleotides may inhibit the polymerase enzymes involved in nucleic acid biosynthesis. The recent work of Ts’O and coworker^^+^^ has established that vertical stacking of purine with (14) A. M. Michelson, C. Monny, R. A. Laursen, and N. J. Leonard, Bioohim. Biophyu. Acta, 118, 258 (1988). (15) P. 0. P. Ts’0, I. 6 . Melvin, and A. C . Olsen, J . Amer. Chem. SOC.,86, 1289 (1963). (16) P. 0. P. Ts’O and 8. I. Chan, $bid., 80, 4176 (1984). (17) A. D. Broom. M. P. Schweirer, and P. 0. P. Ts’O.ibid.. 88, 3612 (1967).
1222 STOUT AND ROBINS
The Journal of Organic Chemistry
SCHEME I11
SCHEME IV
0
How OH OH
OSi(CH,), I
+ aanomer
HOqHZ
YP OH
I
OR
I
0
ti
Iv
XVIII, R=-C-CeHACH3-p XIX, R = H “2
I
OH
OAc
XXI
1
xx S
NH, I
ox% I
“
I
OH
XIII
XJY
1I
pCH,C6HI-COCH2
O OAcV
xv
XVI
pyrimidine nucleosides occurs extensively in aqueous solution. It has been recently postulated “that the stacking energy involved in the interaction of the neighboring bases is the major force contributing to the stability of nucleic acid helices.” l 8 The additional stacking energy due to electron interaction of the benzene ring in the pyrimidine nucleosides described in the present work could contribute significantly to greater stability of a nucleic acid molecule into which these nucleosides are incorporated. Such interactions could well be determined by magnetic resonance spectroscopy. The quinazoline nucleosides described may prove to be important tools for future bioch emica1 studies
.
Experimental Section Melting points were determined with a Thomas-Hoover melting point apparatus and are uncorrected. 2,4-Bis(trimethylsilyloxy)quinazoline (11).-2,4-&uinazolinedione’g (I) (50 g, 0.31 mole) was azeotropically dried in 2.4 1. of refluxing toluene for 1.5 hr employing a Dean-Stark trap. The solution was cooled to ca. 60’ and 90 ml of trimethylchlorosilane added. Reflux was again attained and 114 ml of triethylamine (18) W. M. Huang and P. 0. P. Ts’O, J . Mol. B i d . , 16, 523 (1966). (19) N. A. Lange and F. E. Sheibley, “Organic Synthesis,” CoU. Vol. 11. John Wiley and Sons, Inc., New York. N. Y., 1943. p 79.
dissolved in 400 ml of toluene was added dropwise over a 30-min period. The mixture was refluxed with stirring for an additional 6 hr and left a t room temperature overnight. The triethylammonium chloride was removed by filtration and the toluene solution evaporated under reduced pressure. The residue was transferred to a small flask for distillation. The silyl derivative distilled at 93-98’ a t 0.08 mm and a bath temperature of 178’ to yield 78.5 g (83%) of colorless oil. 1-( 2’,3’,5‘-T~-0-benzoyl-p-~-ribofuranosyl)-2,4-q~nazolinedione (111).-2,3,5-Tri-O-benzoyl-~-ribofuranosylbromide from 25 g (0.05 mole) of 1-0-acetyl-2,3,5-tri-O-benzoylribofuranose was dissolved in 350 ml of dry acetonitrile and 15 g (0.05 mole) of 2,4-bis(trimethylsilyloxy)quinazoline(11) was added. The cloudy solution was stirred for 0.5 hr during which time the solution became clear. The reaction was allowed to proceed at room temperature for 50 hr and the solution then evaporated to dryness under reduced pressure. The resulting foam was dissolved in 180 ml of boiling absolute ethanol and 45 ml of water was added. This solution was stirred and heated on the steam bath for 15 min to achieve crystallization of the prodiict. The mixture was cooled and filtered and the product dried to yield 26.9 g (90%) of 111, mp 180-182”. Further recrystallization from 80% ethanol gave fine colorless needles which melted a t 180.5-181.5”. Anal. Calcd for C ~ ~ H M N(mol ~ O wt O 606.56): C, 67.32; H, 4.32; N, 4.62. Found: C, 67.53; H, 4.40; N, 4.31. 1-( p-~-Ribofuranosyl)-2,4-qdnazolinedione (IV). Method A. -1-( 2’,3’,5’-Tr~-0-benzoyl-~-~-r~bofuranosyl)-2,4-qu~nazol~nedione (111) (12 g, 0.02 mole) was added to a solution of 1.15 g of sodium in 250 ml of absolute methanol. The solution was refluxed for 0.5 hr then evaporated to dryness under vacuum. The resulting solid was partitioned between 500 ml of water and 150 ml of ether. The aqueous portion was extracted twice more with 100 ml of ether each and the combined ether extracts were washed with 50 ml of water. The aqueous phases were combined and acidified to pH 5 with approximately 60 ml of Dowex 50 X 2 ( H f ) resin. The resin was removed by filtration and the aqueous phase concentrated to approximately 40 ml. The cooled solution was filtered and the product recrystallized from a mixture of 700 ml of methanol and 40 ml of water which was concentrated to 150 ml and cooled. The product was filtered and dried to yield 5.08 g (88%) of product, mp 226-227”. Method B.-1-( 2’,3’,5’-Tri-0-benzoyl-p-~-ribofuranosyl)-2,4quinszolinedione (111) (10 g, 16.5 mmole) was heated with 150 ml of methanol (previously saturated with ammonia at
VoE. 33, N o . 3, March 1968
SYNTHESIS OF SOMEQUINAZOLINE NUCLEOSIDES 1223
0') in a sealed bomb at 100' for 8 hr. The bomb was cooled titioned between 100 ml of water and 25 ml of ether. The overnight, the methanol solution evaporated to dryness and aqueous phase was extracted with two 10-ml portions of ether the residue triturated with 400 ml of dichloromethane. then acidified to pH 6 with 10% hydrochloric acid. The soluThe resulting solid was recrystallized from a mixture of 500 tion was concentrated to ca. 5 ml and the solid filtered. This ml of methanol and 10 ml of water which was concensolid w t t ~recrystallized twice from ethanol and melted at trated to 250 ml. The product was removed by filtration and 179-180", [ a j Z 8$53" ~ (c 1.0,dimethylformamide). dried to yield 4.43 g (91%) of needles with mp 226-226.5' Anal. Calcd for C13H~aNzO&3 (mol wt 310.32): C, 50.31; H, and [ a I z 8+16' ~ (c 1.0,dimethylformamide). This product was 4.55; N,9.03. Found: C, 49.96; H, 4.88; N,8.69. identical with that prepared by method A. 3-N-Methyl-2,rl-quinazolinedionefrom Methylation of 1V.-1Anal. Calcd for CtsHlaNzOB (mol wt 294.26): C, 53.06; H, (8-D-Ribofuranosyl)-2,4-quinazolinedione (IV)(0.29g , 1 mmole) 4.80; N, 9.52. Found: C, 53.05; H, 4.96; N, 9.37. was stirred in 2 ml of dry dimethylformamide containing 0.1 g 1-(2',3',5'-Tr~-O-benzoyl-p-~-ribofuranosyl)-4-t~o-2-quinazo- of dry potassium carbonate and 0.01 ml of methyl iodide for lone (VI.-To a solution of 6.0g (0.01mole) of l-(2',3f,5'-tri-O- 20 hr a t room temperature. The potassium carbonate was benzoyl-@-~-ribofuranosy1)-2,4-quinazolinedione (111) in 100 ml filtered and washed with 1 ml of dimethylformamide. The of pyridine was added, with stirring, 8.9 g (0.038 mole) of filtrate was diluted with 25 ml of dichloromethane and the phosphorus pentasulfide. The solution was refluxed gently resulting solid removed by filtration. This filtrate wm taken to dryness and the residue refluxed in 10 ml of 10% hydrochloric (bath temperature 130-140") for 17 hr. Completion of reaction was determined by tlc on alumina H F 254 plates with acid for 1 hr. The solution was filtered hot and gave tan methanol-ethyl acetate ( I :20) arJ solvent. The pyridine soluneedles on cooling. The needles were recrystallized from tion was evaporated to one-third the original volume and ethanol-water using charcoal. The resulting needles were poured into 2 1. of water a t 60". The mixture was stirred for sublimed for analysis (bath temperature 175-180"), and melted at 240-241'. 2-3 hr then left at room temperature overnight. The resulting solid was filtered, dissolved in chloroform, which was dried Anal. Calcd for C9H8N2O2(mol wt 176.17): C, 61.36; H, and removed under vacuum. The resulting foam was boiled 4.58; N, 15.90. Found: C, 60.82; H, 4.43; N, 16.10. with 150 m! of methanol to induce crystallization. The solid This material on mixture melting point determination with was filtered and dried to yield 5.75 g (93%) of yellow solid authentic material prepared by the method of Steiger and Wagwhich melted a t 18O-1Ylo. Tlc on silicAR 7 G F with chloroner? was undepressed and the ir and uv curves were identical. form-ethyl acetate (9:1) showed ca. 3-5% unconverted 111. 1-( 2',3'-O-Isopropyl~dene-p-~-~bofuranosyl)-2,4-q~nazolineThe analytical sample was prepared by preparative plate dione (IX).-A solution of 450 ml of dry acetone, 1.60ml of 2,2chromatography on silicAR 7GF with the same solvent and V dimethoxypropane, and 2.0 ml of 70% perchloric acid was recrystallized from ethanol-benzene (10:1). The analytical prepared and left at room temperature for 5 min. To this solution was added 1.32g (4.5mmoles) of l-(p-D-ribofuranosyl)sample melted at 179.5-180.5". 2,4-quinazolinedione (IV) and the mixture was stirred for 0.5 hr. .4nal. Calcd for C34H26N208S (mol wt 622.63): C, 65.58; H, 4.21; N, 4.50. Found: C, 65.81; H, 4.47; N,4.66. Pyridine (1.6 ml) was added and the solution was concentrated 4-Amino-l-(@-~-ribofuranosyl)-2-quinazolone (VII). Method to ca. 70 ml where 23 ml of 10% sodium carbonate was added. A.-1-( 2',3f,5'-Tr~-0-benzoyl-~-~-r~bofuranosyl)-4-th~o-2-qu~nThe solution was again concentrated to remove the remaining azolone (Y) (2g) was heated in 150 ml of methanol (previously acetone. The aqueous solution was then extracted four times saturated with ammonia a t 0") in a bomb a t 100" for 7 hr and with dichloromethane (one 25-ml portion and three 10-ml porthen cooled to room temperature overnight. The resulting tions). The combined dichloromethane solution was dried and solution was conrentrated under vacuum. The residue was evaporated. The resulting glass was crystallized by solution in 30 ml of benzene followed by the addition of 20 ml of cyclotriturated with 250 ml of dichloromethane and the resultant hexane to yield 1.43 g (95%) of material which melted a t solid was recrystallized from 180 ml of methanol and 15 ml of 120-123". Crystallization of IX from benzene-cyclohexane water concentrated to ca. 25 ml. The product weighed 0.77 g gave pure product which melted a t 125-127". (82%) and melted at 259-260' dec. Method B.-1-( 2',3',5'-Tri-O-benzoyl-p-~-ribofuranosyl)-2,4Anal. Calcd for CleHlsNzOB(mol wt 334.32): C, 57.48; H, 5.43; N,8.38. Foirnd: C, 58.05; H, 5.80; N, 8.17. quinazolinedione (111) (6g, 0.01mole) was refluxed in a solution 1-(2',3'-O-Isopropylidene-5'-methanesulfonyl-~-~-ribofuranof 60 ml of phosphorus oxychloride and 3 ml of N,N-diethylosyl)-2,4-quinazolinedione(X).--A solution of 0.85 g of I-( 2',3'aniline for 10 min. The phosphorus oxychloride and N,Ndiethylaniline were removed under vacuum and the resulting O-~sopropylidene-p-~-ribofuranosyl)-2,4-quinazolined~one ( IX) and 0.21 ml of methanesulfonyl chloride (1.1 moles) in 15 ml foam was dissolved in 20 ml of chloroform. This solution was poured slowly into a vigorously stirred ice-cold solution of 2% of dry pyridine was left in the refrigerator for 15 hr and then a t room temperature for an additional 7 hr. Water (0.45 ml, sodium hydroxide (200 ml). Dilute sodium hydroxide was 10 moles) was then added and the solution stirred at room added as necessary to keep the p H greater than 1 1 a t all times. temperature for 45 min. At this time 25 ml of water was The basic solution was extracted with two 100-ml and two added and the solution extracted with three 25-m1 portions of 50-ml portions of chloroform. The combined chloroform solution was then washed with ice water and saturated salt solution chloroform. The chloroform was washed with six 10-ml portions of ice-cold 3 N sulfuric acid until the washes were distinctly and dried over sodium sulfate containing a little potassium acidic and then twice with 5% sodium bicarbonate (20ml and carbonate. The yellow solution was evaporated to dryness; 5 ml) until the washes were basic. The chloroform solution the oil was placed in a sealed bomb with 170 ml of methanolic ammonia and heated a t 100' for 7 hr. The cooled methanol was then dried over sodium sulfate and taken to dryness. The foam was crystallized by solution in 8 ml of benzene followed solution was evaporated, the residue triturated with dichloromethane, and the solid, mp 140-180",crystallized from 400 ml by the addition of 1 ml of cyclohexane to yield 0.95g of crystals of methanol and 50 ml of water which was concentrated to ca. which melted at 190-192" with some softening ca. 125". The product was recrystallized from benzene-methanol and melted 80 ml. This solution was ceoled to room temperature and at 190-1 92". filtered. The product was a mixture of 4-amino-l-(p-~-ribofuranosy~)-2-quinazolone(1'11) and l-(p-~-ribofuranosyl)-2,4Anal. Calcd for C1,H20N208S(mol wt 412.41): C, 49.51;H, quinazolinedione (IV). Separation of VI1 from I V was easily 4.89; N,6.79. Found: C, 49.16; H, 4.99; N,6.71. achieved by fractional crystallization since VI1 crystallizes first Oz,5'-Anhydro-l-( 2',3'-O-isopropylidene-p-~-ribofuranosyl)-4quinazolone (XI). Method A.-To a refluxing mixture of 0.4 g from a methanol-water mixture. Yield of the cytidine (0.97 mmole) of l-(2',3'-O-isopropylidene-5'-methanesulfonylanalog VI1 was 1.52 g (52%) and of the uridine analog I V was 0.90 g (31%). One additional recrystallization of VI1 from p-~-ribofuranosyl)-2,4-quinazolinedione and 0.3 g of anhydrous methanol-water gave material which melted a t 259-260" dec ; sodium iodide in 6 ml of dioxane was added six drops of water and an additional 6 ml of dioxane. After 1.5 hr refluxing the [ a I z 8-3" ~ ( c 1.0,dimethylformamide). Anal. Calcd for C ~ S H ~ S N S (mol O ~ wt 293.27): C, 53.24; H, bumping from precipitated salts was such that six more drops 5.16; N, 14.33. Found: C,53.25; H, 5.24; N, 14.33. of water and 2 ml of dioxane were added and reflux continued 1-(~-~-Ribofuranosyl)-4-thio-2-quinazolone (VIII).-A solu1 hr. To the cooled solution was added 50 ml of dichloromethtion of 0.5 g ( 1.7 mmoles) of I-(2',3',5'-tri-O-benzoyl-p-~-riboane and this solution was extracted with three 10-ml portions furanosyl)-4-thio-2-quinazolone(V)and 0.1 g (4.3 g-atoms) of of water. The water was back extracted with 5 ml of dichlorosodium in 15 ml of absolute methanol was refluxed gently for methane and the combined organic phases were washed with 70 min. The methanol was evaporated and the residue parsaturated salt solution and then dried over sodium sulfate.
1224 STOUTAND ROBINS
The Journal of Organic Chemistry
one (XVIII) in 45 ml of pyridine was added 2.1 g (9.4 mmol) The dichloromethane was removed under vacuum and the of phosphorus pentasulfide and the solution gently refluxed resulting oil plus 0.7 g of silver acetate was refluxed for 15 rnin for 13 hr (bath temperature 120-130'). Completion of the in 100 ml of absolute methanol. The precipitated silver reaction was determined by tlc on alumina H F 254 plates iodide was filtered and the filtrate saturated with hydrogen using methanol-ethyl acetate (1:9) as solvent. A t this point sulfide to remove any residual silver. Again the methanol there w&s some unreacted XVIII but also there was some was filtered and the filtrate concentrated to ca. 2 ml. The cleavage of the nucleoside. The solution was reduced by resulting precipitate was recrystallized from 15 ml of benzene ca. two-thirds under vacuum and the remaining pyridine and 0.5 ml of methanol concentrated to 10 ml and then 1 ml of solution poured into 700 ml of water at ca. 45'. The solution, cyclohexane added. The compound began to darken a t 270' after cooling, was extracted seven times with chloroform (one and melted a t 280-285" dec. 100-ml and six 50-ml portions). The chloroform extracts Anal. Calcd for C1BH1BN206(mol wt 316.30): C, 60.75; H, were combined and washed with ice water and saturated salt 5.10; N, 8.86. Found: C, 60.55; H, 5.25; N, 8.88. solution and then dried over sodium sulfate. The chloroform Method B.-A solution of 0.81 g (0.002 mole) of 1-(2',3'-0was removed under vacuum and the residual foam crystallized isopropylidene-5'-O-methanesulfonyl-p-~-r~bofuranosyl) - 2,4by boiling in 50 ml of methanol with vigorous stirring. This quinazolinedione (X) in 30 ml of dry dimethylformamide and yielded 1.22 g (75%) of yellow crystals which melted a t 1944 ml of 0.5 M potassium t-butoxide in t-butyl alcohol was 196'. Purification was achieved by preparative layer chromaheated a t 100" for 1.5 hr. The gellike solution was taken to tography on silicAR 7GF with ethyl acetate-chloroform (1:20) dryness and the residue extracted twice with boiling chloroform. as solvent and the product was crystallized from benzeneThe chloroform was taken to dryness and the residue crystalethanol. The analytical sample melted a t 195.5-196'. lized from 30 ml of benzene and 4 ml of methanol concentrated Anal. Calcd for C29H26N~06S (mol wt 530.58): c, 65.64; to 10 ml. This gave 0.43 g of product (90%) which melted a t H, 4.94; N, 5.28. Found: C, 65.43; H, 5.07; N, 5.31. 280" after softening at 245". This product (XI) was identical 4-Amino-1-(p-~-2'-deoxyribofuranosyl)-2-quinazolone (XXI). with that prepared by method A. 1-(3',5'-Di-O-p-tolyl-p-~-2'-deoxyr~bofuranosyl)-2,4-quinazo--A mixture 0.75 g (1.4 mmoles) of 1-(3',5'-di-O-p-tolyl-p-~-2'deoxyribofuranosyl)-4-thio-2-quinazolone (XX) in 100 ml of linedione (XVIII).-3,5-Di-O-p-tolyl-2-deoxyribofuranosyl chlomethanolic ammonia (saturated a t 0") was left at room temperride" (14 g, 0.035 mole) and 15 g (0.05 mole) of 2,4-bis( trimethylature in a pressure bottle for 3 days. The methanolic solution sily1oxy)quinaeoline (11) were reacted in 300 ml of dry acewas taken to dryness and the residue triturated with 10 ml of tonitrile. The solution remained cloudy throughout the reacdichloromethane and 50 ml of ether. The solid was filtered tion period. After 42 hr the reaction mixture was evaporated and recrystallized from a methanol and water mixture. The to dryness. The trimethylsilyl groups were hydrolyzed by deoxycytidine analog XXI melted at 212.5-213'; [ a l Z s+72" ~ dissolving the product in 100 ml of boiling ethanol and adding ( c 1.O, dimethylformamide). 20 ml of water. This solution was boiled for several minutes Anal. Calcd for CISHI,N~O,.~/,H?O (mol wt 286.28): C, then evaporated to dryness under vacuum. The solid wras 54.54; H, 5.63; N, 14.68. Found: C, 54.19; H, 5.65; N, 14.70. triturated with 500 ml of dichloromethane, filtered, and evap0~,2'-Anhydro-1-(p-~-arabinofuranosy1)-4-quinazolone (XU). orated leaving 16.61 g of mixed deoxynucleosides plus some -A mixture of 3.0 g (1.02 mmoles) of l-(p-~-ribofuranosyl)-2,4sugar. The a and p anomers could be separated by tlc on quinazolinedione (IV), 2.85 g (1.33 mmoles) of diphenyl carsilicAR 7GF using chloroform-ethyl acetate (4: 1). bonate, and 0.05 g of sodium bicarbonate in 10 ml of dry diPurification was achieved by column chromatography on methylformamide was heated a t 150' for 30 min. There was neutral alumina (Merck). The column was 2 in. in diameter an initial vigorous evolution of gas which subsided after 5 min. by 22 in. high and contained 800 g of alumina. The column Tlc on silicAR 7GF with ethyl acetate-methanol ( 9 : l ) as was packed in benzene. The nucleoside mixture was dissolved solvent showed very little starting material after 10 min. The in benzene and applied to the column. The sugar was washed refluxing solution was allowed to cool for a few minutes and through in 550 ml of 20% benzene-ethyl acetate and an addithen poured slowly into 800 ml of vigorously stirred ether. The tional 1100 ml of 20 % benzene-ethyl acetate eluted nothing precipitate was filtered and recrystallized from 200 ml of more. The eluent was changed to ethyl acetate and after 1700 methanol concentrated to 100 ml. The filtered and dried ml of ethyl acetate the p anomer began to be eluted. The solid weighed 2.53 g (90%) and melted a t 259-259.5'. following 4450 ml contained pure anomer which weighed 4.7 g Bnal. Calcd for C13H12N208(mol wt 276.24): C, 56.52; H, on evaporation of the solvent. From this point the a anomer 4.38; N , 10.14. Found: C, 56.56; H, 4.42; N , 10.12. was eluted with the p anomer. 1-(p-~-Arabinofuranosyl)-2,4-quhazolinedione (XIII).-02,2'Total weight of material eluted from the column was 14.9 g Anhydro-1-( ~-~-arabinofuranosyl)-4-quinazolone (XII) ( 1 g) (90%) with 3.8 g of sugar, 4.7 g of pure p anomer (25.4%), was heated on the steam bath in 50 ml of 0.05 M sodium hydrox6.2 g of mixed anomers, and 0.2 g of a anomer. The over-all yield of a and 9, anomers was 60%. ide for 15 min. Completion of the hydrolysis was determined The pure p anomer was recrystallized from benzene-cycloby tlc on silicAR 7GF with water as developing solvent. The solution was cooled and neutralized to p H 5-6 hexane and gave 4.21 g which melted at 126-131'. Further crystallization from the same solvent gave material which with Dowex 50 X 2 (H+). The resin was filtered and the melted a t 129-134'. aqueous solution concentrated t o dryness. The solid was recrystallized from 50 ml of methanol and 7 ml of water conAnal. Calcd for Cd&sNzO, (mol wt 514.41): C, 67.69; H, 5.09; N, 5.45. Found: C, 67.47; H, 5.12; N, 5.45. centrated to 5 ml. The thick colorless rods weighed 1.03 g 1-(~-~-2'-Deoxyribofuranosyl)-2,4-quinazolinedione (XIX).(98%) and melted a t 209-211" but resolidified as it apparently 1-(3',5'-D~-0-p-tolyl-~-~-2'-deoxyr~bofuranosyl)-2,4-quinazo-lost the sugar, [a]% -18" (c 1.0, dimethylformamide). linedione (XVIII) was added to a solution of 0.1 g (4.3 g-atoms) Anal. Calcd for C I ~ H I (mol ~ Nwt~ 294.26): ~ ~ c, 53.06; H, 4.80; N, 9.52. Found: C, 52.99; H, 4.80; N, 9.70. of sodium dissolved in 21 ml of absolute methanol and the 1- (2',3',5'- Tri-0-acetyl-p-D-arabinofuranosyl)-2,4- quinazosolution was allowed to reflux gently for 30 min. After evaporation of the solvent the residue was partitioned between linedione (XIV).-A solution of 0.90 g (3.05 mmoles) of l - ( p - ~ arabinofuranosyl)-2,4-quinazolinedione (XIII) in a mixture of 100 ml of water and 25 ml of ether. After two more extractions with ether the organic phases were combined and back ex10 ml of pyridine and 10 ml of acetic anhydride was left a t tracted with 15 ml of water. The aqueous phases were comroom temperature overnight. The clear solution was poured bined and carefully acidified to pH 5 with 6 ml of Dowex into a well-stirred mixture of ice water (200 ml). The mixture was stirred a t 0" for 2 hr then filtered. The solid was recrystal50 X 2 (H+) resin. The resin was removed by filtration and lized from methanol by the addition of water to give 1.30g the filtrate concentrated to 3 ml, cooled and filtered and the solid dried leaving 0.34 g of colorless needles which melted a t (100%) which melted at 205-206'. 159-164". This material was recrystallized from benzeneAnal. Calcd for ClVH20N20D (mol wt 420.37): C, 54.28; H, methanol to yield 0.26 g (69%) which melted a t 167.5-168', 4.80; N, 6.66. Found: C, 54.40; H, 4.85; N, 6.53. [a]'8D +39" ( c 1.0 dimethylformamide). 1-( 2',3',5'-Tri-O-acety1-p-~-arabinofuranosyl) -4-thio-2-quinazolone ( XV) .-1-( 2',3',5'-Tri-O-acetyl-p-~-arabinofuranosy1)Anal. Calcd for C13HllN206(mol wt 278.26): C, 56.11; H, 5.07; N, 10.07. Found: C, 56.14; H, 5.22; N, 10.23. 2,4-quinazolinedione (XIV) (1.30 g, 3.1 mmoles) was dissolved 1-(3',5'-Di-O-p-tolyl-p-~-2'-deoxyribofuranosyl)-4-thio-2-quinin 50 ml of pyridine and 2.9 g (12.5 mmoles) of phosphorus azolone (XX).-To a solution of 1.57 g (3.05 mmoles) of 1pentasulfide was added. The solution was stirred a t reflux for 7 hr (bath temperature 140"). Completion of the reaction ( 3',5'-di-O-p-tolyl-~-~-2'-deoxyribofuranosyl) -2,4-quinazolinedi-
Vol. 99, No. 9, March 1968
NOTES 1225
was determined by tlc on alumina H F 254 with methanol10 min. The resulting 2-amino-4-quinazolone which was isolated was identical with the product described by Trattner, et ethyl acetate (1:20) as solvent. One-half to two-thirds of al.20 the pyridine was removed under vacuum and the remaining Hydrolysis of 1-(P-~-Arabinofuranosy~)-2,4-quinazdlinedione This solution was poured into 900 ml of water at 65-70'. (XIII).-A solution of 5-10 mg of XI11 in 2 ml of 10% hydrosolution was stirred and cooled as the thione crystallized. The chloric acid was heated on the steam bath for 1hr. The cooled yield was 1.02 g (77%) which melted a t 149-150". 4-Amino-l-(~-~-arabinofuranosyl)-2-quinazolone (XVI).-1solution yielded 2,4-quinazolinedione ( I ) as a precipitate. The filtrate was chromatogrammed against D-ribose, a-deoxy-~(2',3',5'-Tri-O-acetyl-P-~-arabinofuranosyl) -4-thio-2-quinazoribose, D-arabinose, and D-xylose on Whatman No. 1 paper lone (XV) (0.90 g, 2.05 mmoles) was heated in 125 ml of using the ethyl acetate-n-propyl alcohol-water (4: 1:2) system methanolic ammonia a t 100" for 6 hr in a bomb. The reaction described by Ha1l.l' A second chromatogram was run using was cooled and filtered and the filtrate taken to dryness. Crysbutanol saturated with water as solvent. Both chromatotallization of the residue from 60 ml of methanol and 10 ml of grams were run for 40 hr and gave good separation of the known water concentrated to 13 ml gave 0.38 g (63%) of XV. The sugars with the unknown sugar at the same Rr as arabinose. product was crystallized from methanol-water. The melting The sugars were detected using the aniline hydrogen phthalate point was 217-219' dec; [CY]*~D-24' ( c 1.0, dimethylformamide). spray of Partridge.22 Anal. Calcd for ClaHl5NaOs.HzO (mol wt 311.29): C , 50.15; H, 5.50; N, 13.50. Found: C, 50.28; H, 5.43; N, 13.48. 2-Amino-1-(2',3'-O-isopropylidene-~-~-ribofuranosyl)-4-q~n- Registry No.-I, 86-96-4;11, 15135-19-0;111, 15135azolone.-0~,5'-Anhydro-l-( 2',3'-O-isopropylidene-P-~-ribofu- 20-3;IV, 15135-21-4;V, 15180-27-5;VII, 15135-22-5; ranosyl)-4-quinazolone ( X I ) (660 mg) was treated with 50 ml VIII, 15135-23-6;IX,15185-75-8;X,15185-76-7;XI, of liquid ammonia in a sealed bomb a t room temperature for 15135-24-7;XII, 15135-25-8;XIII, 15135-26-9;XIV, 50 hr. After removal of the ammonia the product was crystal15185-77-0;XV,15180-28-6;XVI,15135-27-0;XVIII, lized from benzenemethanol and gave colorless needles which 15135-28-1; XIX,15135-29-2;XX, 15135-30-5;X X I , melted a t 171-172' after sintcring at 168". Anal. Calcd for C16HluNaOa(mol wt 333.34): C, 57.65; H, 15135-31-6; 3-N-methyl-2,4-quinazolinedione1 607-19-2; 5.75; N, 12.61. Found: C , 58.35; HI 5.92; N, 12.33. 2-amino-1-(2',3'-0-isopropylidene fl- D ribofuranosy1)2-Amino-4-quinazolone.-All attempts to remove the isopropylidene group from 2-amino-1-(2',3'-O-isopropy~idene-~-~- 4-quinazolone, 15135-33-8. ribofuranosyl)-4-quinazolone proved futile as the sugar linkage (20) R. B. Trattner, G. B. Elion, G . H. Hitchings, and D. M. Sharefkin. was too labile under the conditions necessary to remove the J . Org. Chem., 49, 2674 (1964). isopropylidene group. The nucleoside (200 mg) was hydrolyzed (21) R. H. Hall, Anal. Biochem.. 4, 395 (1962). (22) 9. M. Partridge, Nature, 164, 443 (1949). by heating on the steam bath in 25 ml of 20% formic acid for
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Notes Partition of the ethanol extract of E . dichotoma root bark between ether and 4% hydrochloric acid gave a Voacristine Hydroxyindolenine crude alkaloid fraction upon neutralization of the acid and extraction with ether. The ether extract was H. K. SCHNOES,~ DAVIDW. THO MAS,^ R. AKSORNVITAYA,~ washed with 1% sodium hydroxide to remove the WILLIAMR. SCHLEIGH,~ A N D S. MORRISK U P C H A N ~ phenolic bases and the nonphenolic bases were chromatographed on neutral alumina. Elution with Space Sciences Laboratory, University of California at Berkeley, benzene-chloroform (1 : 1)yielded a fraction which wm Berkeley, California, Department of Chemistry, Massachusetts Institute of Technology, further fractionated by partition chromatography.6 Cambridge, Massachusetts, and Six purple bands were visible on the partition column. Department of Pharmaceutical Chemistry, After elution, the third band yielded heyneanine (I).6 University of Wisconsin, Madison, Wisconsin The fourth band was rechromatographed on a partition column and yielded four bands. Treatment of the Received August 82, 1967 third band with benzene-Skellysolve B gave a crystalline material. Recrystallization from the same Earlier reports have described the isolation from solvent system yielded colorless crystals, C22H2sNz06 Ervatamia dichotoma (Roxb.) Blatter of coronaridine4 (by high resolution mass spectrometry), mp 176-179' and heyneanine.6 We report herewith the isolation dec, -22' (c 0.51,chloroform). The ultraviolet and characterization of voacristine hydroxyindolenine spectrum(A:::H 229.5,268, 291, 300 (sh), 314; 12,380, and its synthesis from voacristine. 4400, 4780, 4410, 3810) and infrared spectrum sug(1) University of California at Berkeley. This work was supported by gested an indole-type alkaloid bearing substituents in Grant No. NsG-101 from the National Aeronautics and Space Administration. the aromatic ring. The infrared spectrum showed (2) Masaaohusetts Institute of Technology. Investigations at M. I. T. broad bands a t 2.80 (w) and 3.10 p (m), indicative of were supported by grants from the National Institutes of Health, GM-01523 and GM-09352. the presence of hydroxyl groups, and a band at (3) University of Wisconsin. The investigation at the University of 5.76 p (s), indicative of the presence of a carbomethoxy Wisconsin was supported by Public Health Service Grant No. HE-02952 group. The nmr spectrum contained a doublet (1 H) from the National Heart Institute. (4) 5. M. Kupchan, A. Bright, and E. Macko, J . Pharm. Sei., 64, 598 centered a t T 2.66 ( J o r f h o = 8 cps), a doublet of
The Isolation and Structural Elucidation of
(1963). (5) S. M. Kupchan. J. M. Cassady, and S. A. Telang, Tetrahedron Lett., 1251 (1966).
(6) K. 9. Brown, Jr., and S. M. Kupchan, J . Chromatop., 9,71 (lQ62).
